Dna polymerases

Abstract

The present invention relates to a variant archaeal DNA polymerase comprising a modified amino acid sequence of a wild-type amino acid sequence, wherein during DNA replication, the variant polymerase is adapted to mis-incorporate a greater number of incorrect bases than compared to a wild-type archaeal DNA polymerase. Such a variant DNA polymerase may be usefully employed in biological assay systems, such as PCR.

1 . A variant archaeal DNA polymerase comprising a modified amino acid sequence of a wild-type amino acid sequence, wherein during DNA replication, the variant polymerase is adapted to mis-incorporate a greater number of incorrect bases than compared to a wild-type archaeal DNA polymerase.  
   
   
       2 . The variant archaeal DNA polymerase according to  claim 1 , wherein the variant is derived from any one of the archaeal family DNA polymerases illustrated in  FIG. 1 .  
   
   
       3 . The variant archaeal DNA polymerase according to  claim 1 , wherein the variant is derived from an archaeal family B DNA polymerase.  
   
   
       4 . The variant archaeal DNA polymerase according to  claim 1 , wherein the variant polymerase is derived from a family independently selected from a group consisting of:  Eutyarchaeota; Crenarclzaeota ; and  Nanoarchaeota.    
   
   
       5 . The variant archaeal DNA polymerase according to  claim 1 , wherein the variant polymerase is derived from a genus independently selected from a group consisting of:  Thermococci; Methanococci; Thermoplasmata; Methanobacteria; Archaeoglobi; Methanopyrii; Thermoprotei ; and  Nanoarchaeum.    
   
   
       6 . The variant archaeal DNA polymerase according to  claim 1 , wherein the variant polymerase is derived from a species independently selected from a group consisting of:  Pyrococcus  furiosus (NP577941);  Pyrococcus woesei  (P61876);  Pyrococcus  ST700 (CAC12847);  Thermococcus  9°N-7 (1QHTA);  Thermococcus gorgonarius  (P56689);  Thermococcus fumicolans  (AA93738);  Pyrococcus horikoshii  (C71210);  Pyrococcus abysii  (CAA90888);  Pyrococcus  GE23 (CAA90887);  Pyrococcus kodakaraensis  (KOD1)(1GCXA);  Thermococcus  GE8 (CAC12850);  Thermococcus hydrothermalis  (CAC 18555);  Pyrococcus  GB-D (deep vent)(AAA67131);  Pyrococcus glycovorans  (CAB81809);  Thermococcus litoralis  (vent) (P30317); and  Thermococcus  TY (CAA73475).  
   
   
       7 . The variant archaeal DNA polymerase according to  claim 1  having a modified amino acid sequence of wildtype  Pyrococcus furiosus  DNA polymerase (Pfu-Pol).  
   
   
       8 . The variant archaeal DNA polymerase according to  claim 1  having at least one modification in amino acid residues equivalent to the residues forming a loop (or hinge region) between two alpha helices, known as N alpha helix and 0 alpha helix of  Pyrococcus furiosus  DNA polymerase.  
   
   
       9 . The variant archaeal DNA polymerase according to  claim 1 , wherein the variant comprises at least one modification in amino acids equivalent to 471, 472, and/or 473 of  Pyrococcus furiosus  DNA polymerase.  
   
   
       10 . The variant archaeal DNA polymerase according to  claim 9 , wherein the modification at amino residue 471, 472 or 473 comprises a substitution with a small or constraining amino acid residue.  
   
   
       11 . The variant archaeal DNA polymerase according to  claim 10 , wherein the amino acid may include glycine, alanine, proline, or serine.  
   
   
       12 . The variant archaeal DNA polymerase according to  claim 1  with a mutation at amino acid 471.  
   
   
       13 . The variant archaeal DNA polymerase according to  claim 12 , wherein the mutation is T471G or T471A.  
   
   
       14 . The variant archaeal DNA polymerase according to  claim 1  with a mutation at amino acid 472.  
   
   
       15 . The variant archaeal DNA polymerase according to  claim 14 , wherein the mutation is Q472G, Q472A or Q472P.  
   
   
       16 . The variant archaeal DNA polymerase according to  claim 1  with a mutation at amino acid 473.  
   
   
       17 . The variant archaeal DNA polymerase according to  claim 16  wherein the modification is D473G or D473A.  
   
   
       18 . The variant archaeal DNA polymerase according to  claim 1  with a mutation at amino acid 215.  
   
   
       19 . The variant archaeal DNA polymerase according to  claim 18 , wherein the modification is D215A.  
   
   
       20 . A nucleic acid molecule encoding an archaeal DNA polymerase according to  claim 1 .  
   
   
       21 . A method of using a variant archaeal DNA polymerase according to  claim 1 , in random mutagenesis protocols.  
   
   
       22 . A method of causing random mutations in DNA comprising the steps of: 
 (i) denaturing a double strand of DNA by heating a solution containing the DNA, free oligonucleotides, primers and a variant archaeal DNA polymerase according to  claim 1;     (ii) reducing the temperature of the solution to effect annealing of the primer and the DNA; and    (iii) allowing extension of the DNA strand by the variant polymerase.    
   
   
       23 . A kit for random mutagenesis protocols comprising a variant archaeal DNA polymerase according to  claim 1;  and optionally buffers and free bases.  
   
   
       24 . A variant DNA polymerase comprising a modified amino acid sequence of a wild-type amino acid sequence, the variant comprising at least one modification in amino acids equivalent to residues 471, 472, or 473 of  Pyrococcus furiosus  DNA polymerase, wherein during DNA replication, the variant polymerase is adapted to mis-incorporate a greater number of incorrect bases than compared to a wild-type DNA polymerase.  
   
   
       25 . The variant DNA polymerase according to  claim 24 , wherein the DNA polymerase is a DNA polymerase delta (S).  
   
   
       26 . The variant DNA polymerase according to  claim 24 , wherein the variant DNA polymerase is derived from a eukaryote.  
   
   
       27 . The variant DNA polymerase according to  claim 26 , wherein the variant DNA polymerase is derived from a mammal or fungus.  
   
   
       28 . The variant DNA polymerase according to  claim 25 , wherein the variant DNA polymerase is derived from a eukaryote.